Antimicrobial properties are desired in a wide range of materials including those in medical devices, personal care items, surfaces related to food handling, and locations such as hospitals and nursing homes where people have lowered resistance to infection. For use in such applications, antimicrobial peptides must be produced on a large scale.
Although short (<20 amino acid) peptides can be produced in high yields via chemical synthesis (Merrifield, R. B., J. Am. Chem. Soc., 85:2149-2154 (1993)), recombinant production offers the potential for large scale production at a more reasonable cost. However, the expression of short polypeptide chains can be problematic in microbial systems because small peptides are often proteolyzed by the host cell's protein regeneration systems. The expression of antimicrobial peptides is often even more problematic because the peptides can be toxic to the production host cells, leading to limited production and cell death (S. Gottesman, Methods in Enzymology 185:119-129 (1990); Goff and Goldberg, in Maximizing Gene Expression p 287-314, (1986)). Fusion of peptides to larger carrier proteins, which serve as anionic partners can eliminate toxicity, provide stability, and provide a method of affinity purification (K. Terpe, Appl. Microbiol. Biotechnol. Vol. 60: 523-533 (2003)). However, even when the toxic peptide is part of a larger fusion protein, high concentrations of fusion peptide in a cell can lead to toxic effects.
Toxicity and proteolytic degradation have been avoided by using a fusion partner, which promotes aggregation of the fusion protein into insoluble inclusion bodies. Inclusion bodies protect against proteolysis and serve as a purification substrate (T. Kempe et al., Gene v39:239-245 (1985)). Alternatively, carrier proteins designed to direct secretion of the fusion proteins into the medium, allowing recovery of the target fusion proteins directly from the medium, have also been used to overcome production issues. For example, U.S. Pat. No. 5,851,802 describes a series of recombinant peptide expression vectors for peptide fusion secretion. A disadvantage of fusion systems is that they generally require costly cleavage reagents and affinity columns for purification.
Periplasmic targeting sequences have been used to direct heterologous proteins into the periplasmic space of E. coli (Groch, et al., Gene v124: 99-103 (1993)) or into the medium using the cell's secretion system (Cols et al., J. of Molec. Microbiol. and Biotech. v3:507-512 (2001); WO 8910971).
There remains a need for a production expression system for antimicrobial peptides, which avoids toxicity and proteolytic degradation, as well as allows for simple, inexpensive recovery of the peptide.